Immunity
Article
IL-22+CD4+ T Cells Promote Colorectal CancerStemness via STAT3 Transcription Factor Activationand Induction of the Methyltransferase DOT1LIlona Kryczek,1,10 Yanwei Lin,1,2,10 Nisha Nagarsheth,1,3,10 Dongjun Peng,1 Lili Zhao,4 Ende Zhao,1 Linda Vatan,1
Wojciech Szeliga,1 Yali Dou,5 Scott Owens,5 Witold Zgodzinski,6 Marek Majewski,6 Grzegorz Wallner,6 Jingyuan Fang,2
Emina Huang,7,11 and Weiping Zou1,3,8,9,*1Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA2Division of Gastroenterology and Hepatology, Renji Hospital, School of Medicine, Shanghai Jiao-Tong University, Shanghai 200001, China3Graduate Program in Immunology4Department of Biostatistics5Department of PathologyUniversity of Michigan School of Medicine, Ann Arbor, MI 48109, USA6The Second Department of General Surgery, Medical University in Lublin, Lublin 20-081, Poland7Department of Surgery, University of Florida, Gainesville, FL 32610, USA8Graduate Program in Tumor Biology9The University of Michigan Comprehensive Cancer Center
University of Michigan School of Medicine, Ann Arbor, MI 48109, USA10Co-first authors11Present address: Department of Stem Cell Biology & Regenerative Medicine, Department of Colorectal Surgery, Cleveland Clinic,9500 Euclid Avenue, Cleveland, OH 44195, USA
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.immuni.2014.03.010
SUMMARY
Little is known about how the immune system im-pacts human colorectal cancer invasiveness andstemness. Here we detected interleukin-22 (IL-22)in patient colorectal cancer tissues that was pro-duced predominantly by CD4+ T cells. In a mousemodel, migration of these cells into the colon cancermicroenvironment required the chemokine receptorCCR6 and its ligand CCL20. IL-22 acted on cancercells to promote activation of the transcription fac-tor STAT3 and expression of the histone 3 lysine79 (H3K79) methytransferase DOT1L. The DOT1Lcomplex induced the core stem cell genes NANOG,SOX2, and Pou5F1, resulting in increased cancerstemness and tumorigenic potential. Furthermore,high DOT1L expression and H3K79me2 in colo-rectal cancer tissues was a predictor of poor patientsurvival. Thus, IL-22+ cells promote colon cancerstemness via regulation of stemness genes thatnegatively affects patient outcome. Efforts to targetthis networkmight be a strategy in treating colorectalcancer patients.
INTRODUCTION
The interaction between tumor cells and immune elementsmight
directly promote tumor development and progression (Ben-
Neriah and Karin, 2011; Coussens et al., 2013), and/or result in
772 Immunity 40, 772–784, May 15, 2014 ª2014 Elsevier Inc.
immunoediting of the tumor that molds the cancer into either a
dormant state (Dunn et al., 2002; Matsushita et al., 2012) or fos-
ters tumor immune evasion (Pardoll, 2012; Zou, 2005). The role
of tumor-infiltrating CD8+ T cells and regulatory T (Treg) cells
(Curiel et al., 2004; Galon et al., 2006) has been extensively stud-
ied in human cancers. Interleukin-22+ (IL-22+) immune cells are
identified in humans and include both IL-22+CD4+ T (Th22) cells
(Duhen et al., 2009; Trifari et al., 2009) and IL-22 expressing
innate leukocytes (ILC22) (Cella et al., 2009;Spits and Cupedo,
2012), but the role of IL-22+ immune cells is poorly defined in
the human cancer microenvironment.
IL-22-producing immune cells could have a role in molding
cancer, particularly colon cancer. The cytokine IL-22 has been
shown to protect intestinal epithelial cells from bacterial infection
and inflammation damage in mice (Aujla et al., 2008; Basu et al.,
2012; Hanash et al., 2012; Pickert et al., 2009; Sonnenberg et al.,
2012; Sonnenberg et al., 2010; Zheng et al., 2008) and supports
thymic repair (Dudakov et al., 2012). Recent mouse studies
have revealed that IL-22+ cells stimulate tumor cell proliferation
in a bacteria-induced colon cancer model (Kirchberger et al.,
2013), and IL-22 binding protein (IL-22BP) reduces chemical
carcinogen-induced colon-cancer development (Huber et al.,
2012). Interestingly, IL22 polymorphisms might be associated
with an increased risk of colon carcinoma development (Thomp-
son et al., 2010). These data suggest a potential link between
IL-22+ cells and colorectal cancer development and progression
in humans. However, the nature and clinical relevance of IL-22+
cells is poorly defined in patients with colorectal cancer. It is
not known whether and how IL-22+ cells impact human colon
cancer.
It has been demonstrated that cancer-initiating cells or cancer
stem cells play an important role in shaping the invasive cancer
Immunity
IL-22+ Cells Enhance Cancer Stemness
phenotype by contributing to tumor initiation, metastasis/
relapse, and therapeutic resistance (Brabletz et al., 2005; Dean
et al., 2005; Pardal et al., 2003; Reya et al., 2001; Vermeulen
et al., 2012). The key issue in cancer stem cell biology is under-
standing the mechanisms that control cancer cell self-renewal
and expansion. Recent evidence suggests some degree of
external control from the microenvironment that defines the
stem cell niche (Bendall et al., 2007; Cui et al., 2013; Scadden,
2006). Given that the protective role of IL-22 in epithelial cells
(Aujla et al., 2008; Basu et al., 2012; Dudakov et al., 2012;
Hanash et al., 2012; Pickert et al., 2009; Zheng et al., 2008)
and its effects on bacteria (Huber et al., 2012) and chemical
carcinogen (Kirchberger et al., 2013) induced cancer in mice,
we hypothesized that colon cancer-infiltrating IL-22+ immune
cells contribute to cancer stem cell renewal and expansion,
reshape the tumor invasive phenotype, and affect colon cancer
patient outcomes. In this work, we focused on the interaction
between IL-22+ immune cells and cancer (stem) cells. We
demonstrated that IL-22+CD4+ T cells promote colorectal can-
cer stemness via STAT3 transcription factor activation and in-
duction of the methyltransferase DOT1L and that this is relevant
for outcome in patients with colon cancer.
RESULTS
IL-22 in the Tumor Environment Promotes Colon CancerStemnessAs IL-22protects intestinal stemcells from immune-mediated tis-
sue damage in mice (Hanash et al., 2012), we hypothesized that
IL-22+ cellsmight support cancer stemness in patientswith colon
cancer. High amounts of IL-22 mRNA were detected in primary
colon cancer tissues compared to peripheral blood and colon tis-
sue adjacent to the cancer (Figure 1A). Next we examined the
potential effects of endogenous IL-22onprimary tumor formation
in a female NOD Shi-scid IL-2Rgnull (NSG) immune-deficient
mouse model (Cui et al., 2013; Curiel et al., 2004; Kryczek et al.,
2012; Kryczek et al., 2011). To this end, single cell suspensions
were made from fresh human colon cancer tissues. These cells
contained all the primary cellular components in the colon cancer
environment includingCD3+ T cellswithin theCD45+ immune cell
population, and lin�CD34�CD45�FSChiSSChi primary colon can-
cer cells (see Figure S1A available online).We equally divided this
primary colon cancer tissue into twogroups and injected the cells
into NSGmice with a one-time treatment of either anti-human IL-
22monoclonal antibody (mAb) or isotypemAb. Anti-human IL-22
mAb dramatically reduced primary tumor volume (Figure 1B) and
delayed tumor development (Figure 1C) and increased mouse
survival (Figure 1D). Furthermore, we found that grafted colon
cancer tissues (isolated fromNSGmice) (Figure S1B) and original
human colon cancer tissues (Figure 1A) and activated human pe-
ripheral mononuclear cells (PBMCs) expressed human IL-22, but
notmouse IL-22 (Figure S1B). The data demonstrate that human,
but not mouse, IL-22, in the human colon cancer environment
promotes tumorigenesis in the NSG model in vivo.
To confirm the tumorigenic potential of endogenous IL-22, we
injected different concentrations of a colorectal adenocarcinoma
cancer cell line, DLD-1 cells, into NSG mice to determine a non-
tumorigenic concentration. We found that 105 DLD-1 cells failed
to form a tumor in the NSG mouse. However, exogenous IL-22
administration enabled tumor formation with 105 DLD-1 cells
as shown by increased tumor volume (Figure 1E), accelerated
tumor development (Figure 1F), and decreased mouse survival
(Figure 1G). When 106 DLD-1 cells were inoculated into mice,
IL-22 promoted tumor development (Figure S1C) and growth
as well (Figure S1D). Thus, IL-22 might enhance tumorigenesis
by altering cancer stem cell properties.
In support of this notion, IL-22 promoted tumor sphere forma-
tion in a dose-dependent manner (Figures 1H and 1I) and
increased aldehyde dehydrogenase (ALDH1) activity in DLD-1,
HT29, and two primary colon cancer cell lines (Figure 1J).
ALDH1 is an operative marker of human cancer stem cells (Car-
pentino et al., 2009; Kryczek et al., 2012). Furthermore, IL-22
enhanced mRNA (Figure 1K) and protein (Figures 1L and 1M)
expression of multiple core stem cell genes including NANOG,
SOX2, and POU5F1 (OCT3/4), but had no effect on b-catenin
and Wnt signaling (Figure S1E). Altogether, IL-22 stimulates
expression of genes associated with core cancer stemness
and promotes colon tumorigenicity.
IL-22+ Cells Are Recruited into the Tumor and PromoteCancer Stemness via IL-22Given that IL-22 promotes colon cancer stemness, we examined
the cellular source of IL-22 and the phenotype of IL-22+ cells in
the human colon cancer environment. Real-time PCR revealed
that IL-22 was expressed by CD45+ immune cells in the colon
cancer environment (Figure 2A). Based on polychromatic flow
cytometry analysis, CD3+ T cells were found to be the predomi-
nant cell type among CD45+ cells in the colon cancer microenvi-
ronment (Figures S2A and S2B). To further define the pheno-
type of the IL-22+ cells, we sorted colon cancer associated
CD45+ cells into four populations: lineage negative cells (lin�),CD33�CD3�CD56+ cells (with natural killer [NK] or potentially
ILC), CD33+ myeloid cells, and CD3+ T cells. We found that IL-
22 mRNA expression was confined to CD3+ T cells (Figure 2B).
Furthermore, sorted colon cancer-associated CD45+CD3+
T cells, but not colon-associated CD45+CD3� cells or blood
CD45+CD3� cells, spontaneously released IL-22 (Figures
2C and 2D). We analyzed the cytokine profile of IL-22+ cells
in the colon cancer. We found that IL-22+ cells were also
CD3+CD8�CD4+ and expressed the transcription factor RORg.
Of the CD3+CD8�CD4+IL-22+ cells, 30% expressed IL-17, and
whereas IL-4, IL-2, and interferon-g (IFN-g) expression was de-
tected in less than 5%of cells (Figure 2E). Thus, IL-22 is predom-
inantly expressed by CD4+ T cells in the colorectal cancer
microenvironment.
We next examined how peripheral blood IL-22+CD4+ T cells
traffic into the colon cancer microenvironment. We analyzed
the expression of cell trafficking-associated molecules including
chemokine receptors and integrins on IL-22+CD4+ T cells in
blood and colon cancer. We found that IL-22 was expressed
by memory, but not naive, CD4+ T cells in blood (Figures S2C
and S2D). We sorted blood CD4+ T cells into C-C chemokine
receptor type 6 (CCR6)� and CCR6+ populations, and subse-
quently examined IL-22 expression. We did not perform intra-
cellular staining because this affects the detection of surface
antigens. We found that CCR6+, but not CCR6�, cells expressedhigh amounts of IL-22 mRNA (Figure 2F) and protein (Figure 2G).
CCR6+ T cells largely coexpressed CD49D integrin, the ligand
Immunity 40, 772–784, May 15, 2014 ª2014 Elsevier Inc. 773
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45 50 55 60 65 70Tu
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NANOGSOX2
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Control Sox2
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J
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Cell linesIL-22Control
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IL22 expression (x 10-3)
Colon cancer
0 50 100 25 75
Blood
Adjacenttissue
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(legend on next page)
Immunity
IL-22+ Cells Enhance Cancer Stemness
774 Immunity 40, 772–784, May 15, 2014 ª2014 Elsevier Inc.
Immunity
IL-22+ Cells Enhance Cancer Stemness
for VCAM1 (Figure S2E). Among CCR6+ cells, IL-22 was pre-
dominantly expressed by primary (Figure S2F) and activated
(Figure S2G) CD49D+ cells. Thus, IL-22+ cells are enriched in
the CCR6+CD49D+ memory T cell pool.
We then asked whether IL-22+ T cells could migrate toward
primary tumor tissues through chemokine (C-C motif) ligand 20
(CCL20), the ligand for CCR6.We observed that T cells efficiently
migrated in response to CCL20 (Figure 2H), and that the
migrating cells were enriched for IL-22+CD4+ T cells (Figure 2I),
expressing CCR6 and CD49D (Figure 2H). We conducted an
in vivo trafficking assay. CCR6+CD49+ T cells were treated
with neutralizing anti-CCR6 mAb or isotype and were transfused
into human colon cancer bearing NSG mice. After 48 hr, human
T cells migrated into the grafted human colon cancer in NSG
mice (Figures S2H and S2I). These T cells expressed IL-22,
and CCR6 blockade reduced their migration (Figures S2H and
S2I). Furthermore, high amounts of CCL20 (Figure S2J) and
VCAM1 (Figure S2K) were detected in colon cancer tissues.
Human T cells were found in the grafted human colon cancer
in NSG mice (Figure S2L). The data suggests that CCR6 and
CD49D signaling mediates homing of IL-22+CD4+ cells into the
colon cancer microenvironment.
We further investigated the potential effects of primary colon
cancer associated IL-22+CD4+ cells on colon cancer stemness.
To this end, colon cancer cell sphere assay was performed with
autologous colon cancer associated CD4+ T cells. We showed
that these T cells enhanced primary colon cancer cell-sphere
formation, whereas anti-IL-22 abrogated this effect (Figure 2J).
In line with this, these T cells also increased core stem cell
gene expression (Figure 2K) and ALDH1 activity (Figure 2L) in
colon cancer cells. This increase in stemness was reduced
with IL-22 blockade (Figures 2K and 2L). Thus, IL-22+CD4+ cells
traffic into the tumor, and promote colon cancer stemness via
secreting IL-22 in the colon cancer microenvironment.
IL-22 Promotes Colon Cancer Stemness via STAT3ActivationNext, we dissected the molecular mechanisms by which IL-22
promotes colon cancer stemness. Signal transducer and acti-
vator of transcription 3 (STAT3) plays a key role in the crosstalk
between cancer and immune cells in the tumor microenviron-
ment (Lee et al., 2009; Yu et al., 2007). In line with previous
reports (Lejeune et al., 2002; Pickert et al., 2009), we observed
that IL-22 activated STAT3 in colon cancer cells (Figure 3A). We
next examined whether the effect of IL-22 on colon cancer stem-
Figure 1. IL-22 in the Tumor Microenvironment Promotes Colon Cance
(A) IL22mRNAwas detected by real-time PCR in colon cancer tissues, adjacent ti
20 colon cancer patients.
(B–D) Single cells isolated from colon cancer tissue were mixed with anti-IL-22 an
growth (B, *p < 0.05, n = 5 per group), incidence (C, p = 0.037, n = 5 per group),
(E–G) DLD-1 colon cancer cells (105) were preincubated with IL-22 (20 ng/ml) for 1
n = 5 per group, incidence), incidence (F, p = 0.003, n = 5 per group), and anima
(H and I) DLD-1 colon cancer cells were cultured with IL-22. Sphere assay was p
numbers of spheres (I) are shown. *p < 0.05, n = 5.
(J) Colon cancer cell lines (DLD-1 and HT29) and two primary colon cancer cells (1
based on aldefluor fluorescence. Cells treated with DEAB inhibitor were negativ
(p < 0.05 for all, n = 3 repeats per group).
(K–M) DLD-1 colon cancer cells were cultured with IL-22 (20 ng/ml). The mRNA
detected by immunoblotting (L, M). (*p < 0.05, n = 5).
nesswasSTAT3-dependent. To this end,wemanipulatedSTAT3
expression either with specific STAT3 knockdown (sh-STAT3)
(Figure S3A) or forced expression of a STAT3 active domain
(STAT3C) (Figure S3B) in colon cancer cells. Sh-STAT3 resulted
in reduced colon cancer sphere numbers (Figures 3B and 3C).
In the absence of IL-22, expression of STAT3C had minimal
effects on colon cancer sphere formation, but addition of IL-22
increased colon cancer sphere formation in STAT3C-expressing
cells compared with controls (Figure 3D). To determine whether
the effect of IL-22 was relatively STAT3-specific, we additionally
evaluated the role of Notch on colon cancer sphere formation. IL-
22 induced potent and equal sphere formation in colon cancer
cells expressing vectors encoding: GFP (scramble), Notch active
domain (Notch-IC), and Notch-negative domain (Notch-DN)
(Wang et al., 2011; Yamamoto et al., 2001) (Figure S3C). The
data suggest that IL-22-mediated STAT3 activation is necessary
and relatively specific in promoting colon cancer stemness.How-
ever, in the absence of IL-22, forced STAT3 activation alone is
insufficient to strongly induce cancer stemness (Figure 3D).
Consistent with this, IL-6 and IL-17 activate STAT3 but failed to
promote colon cancer sphere formation (Figure S3D).
We further explored the effects of IL-22-activated STAT3
in expression of stemness-related genes. STAT3 knockdown
reduced core stem cell gene expression (Figures 3E–3G). We
speculated that STAT3 might directly bind to the promoters of
core stem cell genes and subsequently induce their expression.
We foundseveral predictions for STAT3bindingon theSOX2pro-
moter region (http://www.sabiosciences.com/chipqpcrsearch.
php). Chromatin immunoprecipitation (ChIP) demonstrated that
IL-22 increased STAT3 binding in several sites on the SOX2 pro-
moter area (Figures 3H and 3I) and suggests that STAT3 might
directly activate stemness genes. STAT3 has been found to
recruit p300 (Nakashima et al., 1999), enhancing target gene
expression. We observed that IL-22 enhanced the binding of
p300 to the SOX2 promoter (Figure S3H). As a positive control,
we used FOS, which has been shown to be bound and activated
by STAT3 (Figures S3E and S3G) (Yang et al., 2003). Thus, IL-22
promotes colon cancer stemness via STAT3 activation and its
associated signaling genes.
DOT1L Regulates IL-22 Dependent Colon CancerStemness via H3K79 MethylationEpigenetic modifications of chromatin and their crosstalk with
transcription factors play an important role in the regulation of
gene expression. STAT3 binding to the promoters of core
r Stemness
ssues, and peripheral blood. *p < 0.05 compared to blood and adjacent tissues,
tibody or control mAb, and then subcutaneously injected to NSG mice. Tumor
and animal survival (D, p = 0.013, n = 5 per group) are shown.
hr, and then subcutaneously injected to NSGmice. Tumor growth (E, *p < 0.05,
l survival (G, p = 0.013, n = 5 per group) are shown.
erformed with 2,000 cells. Representative image of spheres (H) and the mean
, 2) were cultured with IL-22 for 24 hr. ALDH1 activity was determined by FACS
e controls. Results are expressed as fold changes of aldefluor fluorescence.
of stem cell core gene was detected by real-time PCR (K) and proteins were
Immunity 40, 772–784, May 15, 2014 ª2014 Elsevier Inc. 775
IL22
expr
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10-3
)
CD45- CD45+0 24 6 8
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Colon cancer
T cells + anti-IL-22T cells
Control
IL22 expression (x 10-3)
CD33+
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Blood
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migration10 9
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*
Figure 2. IL-22+CD4+ T Cells Traffic into the Tumor via CCR6-CCL20 and Promote Cancer Stemenss via IL-22
(A and B) Different cell populations were sorted from colon cancer tissues and IL-22 expression was measured by real time PCR (5–10 donors, p < 0.05).
(C) Different CD45+ immune subsets (106/ml) were sorted from colon cancer and blood, and cultured for 12 hr. IL-22 was detected via ELISA. (n = 5, *p < 0.05).
(D) CD45+ subsets (106/ml) were sorted from colon cancer and blood, and were activated with anti-CD3 and anti-CD28 for 2 days. IL-22 was detected by ELISA
(n = 5, *p < 0.05).
(E) Single colon cancer environmental cells were stained with anti-CD3, CD8, CD45, RORgc, IL-2, IL-4, IL-17, IL-22, and IFN-g antibodies. The phenotype and the
expression of indicated cytokines were analyzed by FACS. Right panels were gated on IL-22+CD45+ cells. One of three independent experiments is shown.
(F and G) CD4+ T cells were sorted based on CCR6 expression and activated with anti-CD3 and anti-CD28 and antigen-presenting cells. IL-22 mRNA was
detected by real-time PCR (F) and IL-22 protein by ELISA (G) (5 different donors, p < 0.05).
(legend continued on next page)
Immunity
IL-22+ Cells Enhance Cancer Stemness
776 Immunity 40, 772–784, May 15, 2014 ª2014 Elsevier Inc.
p-STAT3 (Y705)
0 15 60
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STAT3
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E
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**
β
Figure 3. IL-22 Promotes Colon Cancer Stemness via STAT3 Activation
(A) Colon cancer cells were treatedwith IL-22 for different time points. The amount of phosphorylated STAT3 and STAT3 protein was detected by immunoblotting.
(B–D) Sphere assay was performed with shSTAT3 (B and C) or STAT3C (D) expressing colon cancer cells in the presence of IL-22. Results are shown as sphere
images (B) and the mean numbers of spheres in triplicate (C and D). (n = 5. *p < 0.01).
(E–G) Colon cancer cells expressing shSTAT3 or scrambled vector were cultured with IL-22. Stem cell core gene mRNAs were detected by real-time PCR after
6 hr (n = 5, *p < 0.05).
(H and I) STAT3-ChIP assay was performed DLD-1 cells cultured with or without IL-22 (mean ± SEM, n = 3, *p < 0.05).
Immunity
IL-22+ Cells Enhance Cancer Stemness
stem cell genes is highly context dependent (Hutchins et al.,
2013) and not all STAT3 activating cytokines induced stemness
(Figure S3D). Thus, STAT3 activation might not completely
(H and I) Migration assay was conducted with CD4+ T cells for 4 hr in the presen
(beforemigration) was analyzed by FACS (H). IL-22 expressionwas quantifiedwith
the mean relative expression (I) (n = 3, p < 0.05).
(J) Sphere assay was performed with autologous colon tumor cells in the presenc
or isotype mAb was added in the assay. Results are shown as the mean numbe
(K and L) Primary colon cancer associated T cells were sorted and activated for 3
the presence of anti-IL-22 or isotype mAbs. The mRNA of stem cell core genes w
FACS after 48 hr (L) (n = 5, *p < 0.05, compared to control and T cells with anti-I
explain the increase in stemness. Because transcription factors
and epigenetic modifications often guide external signals to a
specific genetic response, we wondered whether epigenetic
ce of CCL20. The phenotype of migrated cells, nonmigrated cells, and control
real-time PCR in themigrated and nonmigrated cells. Results are expressed as
e of activated colon cancer-associated T cells in a transwell system. Anti-IL-22
rs of spheres in triplicates (2 of 5 patients are shown. p < 0.01).
days. DLD-1 colon cancer cells were cultured with these T cell supernatants in
as detected by real-time PCR after 6 hr (K) and ALDH activity was detected by
L-22).
Immunity 40, 772–784, May 15, 2014 ª2014 Elsevier Inc. 777
IL-22
- +
H3K9me2
H3K9me3
H3K27me3
H3K79me2
H3
H3K36me3
H4K20me3
Acetyl-H3
A
E
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4
2
1
3
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T1L
AFF
4
Con
trol
MLL
T10
B C D
F
IgGH3K79me2 ControlH3K79me2 IL-22
IgGH3K79me2 ControlH3K79me2 IL-22
H IJ
Inpu
t(%
)
IL-22Control
H3K79me2IgG
0.35
0.30
0.25
0.20
0.15
0
0.10
0.05
IL-22 +EPZ004777
POU5F1 (-9kb)
IL-22Control
H3K79me2IgG
0.14
0.12
0.10
0.08
0.06
0
0.04
0.02
IL-22 +EPZ004777
SOX2 (+6kb)K L
NANOG
IgGH3K79me2 ControlH3K79me2 IL-22
Inpu
t(%
)
0.7
0.5
0
0.3
0.6
0.4
0.20.1
SOX2
Inpu
t(%
)
0.5
0
0.3
0.6
0.4
0.20.1
Inpu
t(%
)
POU5F1
0.5
0
0.3
0.4
0.2
0.1
Sph
eres
(fold
chan
ge)
4.03.53.02.52.01.51.00.50
IL-22Control
*
Sph
ere
num
ber
160140120100806040200
EPZ004777 (µM)100 100
IL-22Control
*
Sph
ere
num
ber
160140120100806040200
EPZ004777 (µM)100 100
IL-22Control
*
Inpu
t(%
)
IL-22Control
H3K79me2IgG
0.14
0.12
0.10
0.08
0.06
0
0.04
0.02
IL-22 +EPZ004777
NANOG (+4kb)
*
* *
Gene expression421 30
DOT1LAFF4
Control
MLLT10
IL-22Control
G
Inpu
t(%
)
M
β-actin
Nanog
Sox2
STAT3
p-STAT3 (Y705)
H3
H3K79me2
(legend on next page)
Immunity
IL-22+ Cells Enhance Cancer Stemness
778 Immunity 40, 772–784, May 15, 2014 ª2014 Elsevier Inc.
Immunity
IL-22+ Cells Enhance Cancer Stemness
control, including histone modifications, is involved in controlling
IL-22-induced stemness gene expression. To this end, we exam-
ined the global changes in several histone marks in IL-22-treated
colon cancer cells. Among several histone marks, we observed
that IL-22 selectively increased the dimethylation of histone 3
lysine79 (H3K79me2) (Figure 4A). Disruptor of telomeric silencing
1-like (DOT1L) is the sole H3K79 methyltransferase (Min
et al., 2003; Ng et al., 2002). EPZ004777 (Daigle et al., 2011; Yu
et al., 2012), a selective inhibitor of DOT1L, reduced H3K79me2
(Figure S4A) and suppressed DLD-1 (Figure 4B) and primary co-
lon cancer (Figure 4C) sphere formation. Several proteins recruit
DOT1L tomediatemethylation of H3K79 includingMCEF (AFF4),
AF9 (MLLT3), and AF10 (MLLT10) (Mohan et al., 2010). IL-22
consistently promoted the expression of DOT1L and AFF4, but
notMLLT10 in DLD-1 (Figure 4D) and primary colon cancer cells
(Figure 4E). To further determine the role of DOT1L in colon can-
cer stemenss, we knocked down DOT1L expression with sh-
DOT1L (Figure S4B) and forced ectopic DOT1L expression
(Figure S4C). Similar to EPZ004777, DOT1L knockdown reduced
colon cancer sphere formation (Figure 4F). Forced DOT1L
expression led to an increase in NANOG (Figure S4D) and
SOX2 (Figure S4E) mRNA in the absence of IL-22. However, IL-
22 further increased stemness gene expression (Figures S4D
andS4E) and sphere numbers (Figure S4F). IL-22 also stimulated
c-Myc expression in colon cancer cells, but DOT1L overexpres-
sion had no effects on IL-22-stimulated c-Myc expression (Fig-
ure S4G). These data suggest that DOT1L signaling activation
potentiates the colon cancer stemness program.
We also explored whether IL-22 regulates H3K79me2 on core
stem cell gene promoters. ChIP assayswith H3K79me2 revealed
an increase in H3K79me2 on the proximal promoter areas of
NANOG (Figure 4G), SOX2 (Figure 4H), and POU5F1 (Figure 4I)
in an IL-22 dependent manner. Moreover, EPZ004777 treatment
resulted in reduced H3K79me2 on the stem cell gene promoter
sites (Figure 4J-L). In support of these observations, IL-22
administration caused the activation of STAT3, H3K79 dimethy-
lation, and core stemness gene activation in human colon cancer
cells in the NSG model in vivo (Figure 4M). These data indicate
that IL-22 regulates colon cancer stemness in a DOT1L- and
H3K79me2-dependent manner.
STAT3 Induces DOT1L Expression and ControlsIL-22-Induced Cancer StemnessBoth STAT3 (Figure 3) and DOT1L-H3K79 signaling (Figure 4) are
involved in the control of IL-22-induced cancer stemness.Wehy-
Figure 4. IL-22 Promotes Colon Cancer Stemness via DOT1L and H3K
(A) Colon cancer cells were treated with IL-22 for 48 hr. Histone modifications w
(B and C) Colon cancer sphere assay was performed in the presence of IL-22 and
(B), primary colon cancer cells (C) (n = 5, p < 0.05).
(D and E) Colon cancer cells were cultured for 12 hr with IL-22. Expression of the
PCR. DLD-1 cells (D), primary colon cancer cells (E) (n = 3, p < 0.05).
(F) Colon cancer sphere assay was performed with colon cancer cells expressing
(n = 5, p < 0.05).
(G–I) H3K79me2-ChIP assay was performed to examine H3K79me2 at the core ste
three experiments is shown.
(J–L) H3K79me2 ChIP was performed to examine occupancy at core stem cell ge
One of three experiments is shown.
(M) IL-22 (0.5ug) was injected into the DLD-1 tumor. After 12–48 hr, tumor tissues
and NANOG proteins. One of three experiments with triplicate sections is shown
pothesized that IL-22-activated STAT3 causes H3K79 methyl-
ation. The ENCODE ChIP-sequence (ChIP-Seq) database
revealed STAT3 binding sites on the promoters of DOT1L,
AFF4,MLLT3, andMLLT10 in a lymphoblastoid cell line (access
number:GSM935557) (FigureS5) (Birney et al., 2007). IL-22 treat-
ment increased STAT3 binding to theDOT1L promoter area (Fig-
ure 5A) and to promoters of two other elements of the DOT1L
complex, AFF4 (Figure 5B) and MLLT3 (Figure 5C), but not
MLLT10 (data not shown). As a confirmatory experiment, we
showed that IL-22 also augmented the binding of p300 to the
promoter area of DOT1L (Figure 5D). Thus, IL-22 promotes the
binding of the transcription factor STAT3 to the promoter area
of the DOT1L complex and controls colon cancer stemness.
In addition to the interaction of STAT3 on the DOT1L promoter,
we explored whether STAT3 could directly impact the amount
of H3K79me2 via DOT1L. Enhanced activation of STAT3 in
STAT3C transduced cells caused a genome-wide increase in
H3K79 dimethylation (Figure 5E), but no increase in sphere for-
mation (Figure 3D) in the absence of IL-22. IL-22 treatment
moderately augmented H3K79 dimethylation (Figure 5E) and
dramatically increased sphere formation (Figure 3D). Accord-
ingly, DOT1L gene expression was increased in colon cancer
cells expressing STAT3C (Figure S3I). Thus, STAT3 activation
could promote H3K79me2 via DOT1L.
To determine whether STAT3 regulates core stem cell gene
expression through DOT1L-dependent H3K79 methylation, we
performed ChIP with H3K79me2 in sh-STAT3 IL-22-treated co-
lon cancer cells. STAT3 knockdown abrogated IL-22-induced
H3K79 methylation on the stem cell core gene promoters of
NANOG, SOX2, and POU5F1 (Figures 5F–5H). The data further
solidify the notion that STAT3 is essential for H3K79 dimethyla-
tion and IL-22 potentiates potent cancer stemness via STAT3
mediated-H3K79 dimethylation. DOT1L knockdown (Figure 5I)
or overexpression (Figure 5J) had no effects on IL-22-mediated
STAT3 activation. This suggests that IL-22-induced STAT3
phosphorylation is DOT1L independent. Altogether, the results
indicate that IL-22-activated STAT3 directly regulates DOT1L
expression and subsequently induces H3K79 methylation at
the stemness genes, facilitating and accelerating stemness
gene activation.
High Amounts of Colon Cancer H3K79-DOT1L PredictPoor Patient SurvivalFinally, we examined the clinical relevance of the IL-22-DOT1L
signaling pathway in colon cancer patients. To this end, we first
79me2
ere analyzed by immunoblotting.
the DOT1L inhibitor EPZ004777. Sphere numbers were recorded. DLD-1 cells
genes encoding members of the DOT1L complex was quantified by real-time
sh-DOT1L or vector in the presence of IL-22. Sphere numbers were recorded
m cell genes promoters in DLD-1 colon cancer cells cultured with IL-22. One of
nes in DLD-1 colon cancer cells cultured with or without IL-22 and EPZ004777.
were extracted for immunoblotting analysis for the STAT3, H3K79me2, SOX2,
.
Immunity 40, 772–784, May 15, 2014 ª2014 Elsevier Inc. 779
Inpu
t(%
)
-0.5 kb
IgGControlIL-22
0.4
0.3
0.2
0.1
0
DOT1L
4.5 kb 7.2 kb 7.5 kb
IgGControlIL-22
AFF4
Inpu
t(%
)
0.04
0.02
0
0.03
0.01
A B C
IgGControlIL-22
0.6
0.4
0.2
0
0.5
0.3
0.1
Inpu
t(%
)Scramble shSTAT3
NANOG (+0.5kb)
F
IgGControlIL-22
MLLT3
0.02
0
0.03
0.01Inpu
t(%
)
IgGControlIL-22
0.6
0.4
0.2
0
0.5
0.3
0.1
Inpu
t(%
)
Scramble shSTAT3SOX2 (+4kb)
IgGControlIL-22
0.8
0.4
0.2
0
0.5
0.3
0.1
Inpu
t(%
)
0.70.6
Scramble shSTAT3POU5F1 (-2kb)
G H
Inpu
t(%
)
-0.5 kb
IgGControlIL-220.008
0.006
0.004
0.002
0
DOT1L
7.5 kb
D
IL-22 0 24Vector
8 hours
p-STAT3 (Y705)
H3K79me2
Flag-STAT3
H3
0 24
STAT3C
8
E
IL-22p-STAT3(Y705)
-Scramble
+ -shDOT1L
+
STAT3
I
J
IL-22p-STAT3(Y705)
0Vector
24 48 0 24 48DOT1L
STAT3
β-actin
*
***
**
**
*
* *
hours
Figure 5. STAT3 Stimulates DOT1L Expres-
sion and Promotes IL-22-Induced Cancer
Stemness
(A–C) STAT3-ChIP assay was performed in DLD-1
colon cancer cells cultured with or without IL-22 to
examine STAT3 occupancy at DOT1L (A), AFF4
(B), and MLLT3 (C) promoters. One of three
experiments is shown.
(D) p300-ChIP assay was performed in DLD-1
colon cancer cells cultured with or without IL-22
to examine p300 occupancy at DOT1L promoter.
(n = 3 with duplicates, *p < 0.05).
(E) DLD-1 cells were transduced with STAT3C
expressing or control lentiviral vectors, and
cultured with IL-22. H3K79me2 was detected
by immunoblotting. One of three experiments is
shown.
(F–H) H3K79me2-ChIP was performed in colon
cancer cells cultured with IL-22 for 24 hr to
examine H3K79me2 occupancy at the promoter
areas of core stem cell genes (n = 3 with dupli-
cates, *p < 0.05).
(I and J) DLD-1 cells were transduced with
shDOT1L (I) or DOT1L expressing (J) or control
lentiviral vectors, and cultured with IL-22. STAT3
and STAT3 phosphorylation were detected by
immunoblotting. One of three experiments is
shown.
Immunity
IL-22+ Cells Enhance Cancer Stemness
analyzed the relationship between IL-22, DOT1L, and stem cell
gene transcripts in patients with colorectal cancer from the
National Center for Biotechnology Information Gene Expression
Omnibus database (GSE17536) (Smith et al., 2010). The
GSE17536 database includes 177 colorectal cancer patients
with clinic and pathological information (Table S1). We found
that IL-22 expression correlated with DOT1L (Figure 6A). More-
over, the expression of DOT1L correlated with SOX2 (Figure 6B).
Furthermore, when we divided patients into ‘‘low’’ and ‘‘high’’
groups based on the median value of SOX2, we observed that
high SOX2 expression was associated with poor patient survival
(Figure 6C). Then, we quantified nuclear DOT1L (Figure S6A) and
H3K79me2 (Figure S6B) via immunohistochemistry in paraffin-
fixed colorectal cancer tissues from patients with available
clinical and pathological information (Table S2). The expression
of DOT1L correlated highly with that of H3K79me2 in the same
tumor (Figure 6D). Furthermore, based on the median values
of DOT1L intensity, we divided patients into ‘‘low’’ and ‘‘high’’
groups (Figure S6A). Overall survival was shorter in patients
with high DOT1L staining compared to low DOT1L expression
(Table S3, Figure 6E). Age and tumor stage (TNM)were important
prognostic factors for colon cancer survival (Table S4). After
adjusting for the clinical factors, overall survival remained shorter
in patients with high DOT1L expression (Table S4). The data
strongly suggest that increased tumor DOT1L abundance is a
780 Immunity 40, 772–784, May 15, 2014 ª2014 Elsevier Inc.
significant and independent predictor of
poor survival in colorectal cancer. We
further analyzed the relationship between
tumor H3K79me2 expression and sur-
vival. Similar results were observed with
H3K79 methylation (Figure S6B; Fig-
ure 6F). Overall survival was shorter in
patients with high H3K79 dimethylation (Table S3; Figure 6F).
Therefore, DOT1L expression and H3K79me2 could be an onco-
genic predictor for poor survival in colorectal cancer. Altogether,
IL-22+CD4+ T cells produce IL-22 and shape colon cancer stem-
ness through a STAT3-DOT1L-mediated stem cell core signaling
pathway.
DISCUSSION
The capacity of immune cells to modulate the cancer phenotype
has been the subject of intensive investigation. The immune sur-
veillance model proposes that immunoediting contributes to
cancer dormancy in a mousemodel (Dunn et al., 2002; Matsush-
ita et al., 2012). It is also well known that immune cell subsets,
when chronically activated, directly foster tumor development
(Ben-Neriah and Karin, 2011; Coussens et al., 2013), and pro-
mote cancer progression (Ben-Neriah and Karin, 2011; Cous-
sens et al., 2013; Cui et al., 2013). As an extension of this model,
we demonstrate that IL-22+ immune cells select and sustain the
invasive phenotype of human colon cancer: cancer stemness.
Th22 cells and IL-22 have been reported to be a protective im-
mune element in infection, inflammation (Aujla et al., 2008; Basu
et al., 2012; Hanash et al., 2012; Pickert et al., 2009; Zheng et al.,
2008), and thymic repair (Dudakov et al., 2012). Colon cancer-
associated IL-22+CD4+ T cells stimulate cancer stem cell core
A
C
-2.7 -2.1 -1.5 -0.9DOT1L
-2.9
-2.8
-2.7
-2.6
-2.5
-2.4
-2.3
-2.2
IL22
-2.7 -2.1 -1.5 -0.9DOT1L
-1.2
-0.9
-0.6
-0.3
0.0
0.3
0.6
0.9
SO
X2
Cum
ulat
ive
Pro
porti
on
Sur
vivi
ng
1.0
0.6
0.4
0.2
00.1
0.3
0.5
0.80.9
0.7
0Time (months)
80 100 120 1406020 40
Low SOX2High SOX2
Cum
ulat
ive
Pro
porti
onS
urvi
ving
1.0
0.6
0.4
0.2
00.1
0.3
0.5
0.80.9
0.7
0Time (months)
40 50 60 703010 20
Low DOT1LHigh DOT1L
B
FE
D
160
145
135
185
155
140
H3K
79m
e2
180175170165
DOT1L
High Low
Cum
ulat
ive
Pro
porti
onS
urvi
ving
1.0
0.6
0.4
0.2
00.1
0.3
0.5
0.80.9
0.7
0Time (months)
40 50 60 703010 20
Low H3K79me2High H3K79me2
*
Figure 6. High Amounts of Colon Cancer
H3K79-DOT1L Predict Poor Patient Survival
(A andB) The correlationbetweenDOT1L, IL22 and
SOX2 transcripts in patients with colorectal cancer
was examined analyzing 177 colorectal cancer
patients (GSE17536). R = 0.31, p = 0.000032 (A);
R = 0.38, p = 0.00000024 (B).
(C) The association between SOX2 transcripts and
patient survival. The analyses were conducted in
177 colon cancer patients (GSE17536), p = 0.046.
(D) The correlation between DOT1L and
H3K79me2 in patients with colon cancer. n = 144,
R = 0.23, p = 0.005.
(E and F) The relationship between tumor DOT1L
(E), H3K79me2 (F) expression and colon cancer
overall survival was evaluated. n = 151, p = 0.001
(E), n = 144, p = 0.039 (F).
Immunity
IL-22+ Cells Enhance Cancer Stemness
gene expression and promote cancer stemness. Cancer stem-
ness and the invasive tumor phenotype are thought to be a
cell-autonomous process specified by the genetic and epige-
netic signature of cancer cells. Our data indicate that IL-
22+CD4+ T cells, a crucial T cell immune component in the
colon cancer microenvironment, functions as an environmental
extrinsic signal, directly targets cancer cells, and defines their
stemness. We focus on patients with advanced colon cancer
due to practical and ethical reasons. Given the defined roles of
IL-22 in cancer stemness, it is possible that ILC22 or/and other
IL-22+ cells employ a similar mechanism and regulate cancer
initiation and development in early phases of colon cancer.
Nonetheless, it is reasonable to conclude that IL-22+CD4+
T cells participate in colon cancer stemness.
STAT3activity is required for small-intestine crypt stemcell sur-
vival in mice (Matthews et al., 2011). We have demonstrated that
both STAT3 activation and DOT1L-H3K79 signaling are essential
Immunity 40, 772–
for IL-22-induced cancer stemness.
STAT3 and DOT1L facilitate core stem
cell gene activation, and are indispensible
in IL-22-induced cancer stemness.
Although understanding the dynamic
interaction between the transcription fac-
tor STAT3 and the histone mark
H3K79me2 warrants further investigation,
our work addresses a central question: do
histone modifications cue and instruct
transcription or support and correlate
withgeneactivity (Henikoff andShilatifard,
2011)? Our findings demonstrate an
instructive role for methylated H3K79 in
colon cancer stem cell gene activation
stimulated by IL-22. Thus, our work re-
veals a relationship between a key tran-
scription factor (STAT3) and an important
epigenetic mark (H3K79) in determining
cancer stemness.
DOT1L is the sole H3K79 methyltrans-
ferase (Min et al., 2003; Ng et al., 2002).
We have shown that IL-22 regulates colon
cancer stemness via DOT1L-H3K79. The
amount of DOT1L expression in colon cancer independently pre-
dicts poor patient survival. Notably, it has been reported that
DOT1L recruitment through Mixed Lineage Leukemia (MLL)
fusion proteins is strongly associated with MLL transformation
(Chang et al., 2010; Jo et al., 2011). It remains to be determined
whether DOT1L is an oncogene in human epithelial cancer, and
whether it is biochemically and functionally linked to the well-
defined colon oncogenes. Nonetheless, our study is the first to
demonstrate that DOT1L contributes to human epithelial carci-
noma, including its involvement in colorectal tumorigenesis
and its regulation by IL-22+CD4+ T cells. Therefore, our work
has revealed epigenetic mechanisms by which IL-22+CD4+
T cells and IL-22 control human colon cancer stemness and
tumorigenesis. Similar mechanisms might apply for other types
of human cancers. Because DOT1L inhibition is a proposed
strategy for targeted therapy of leukemia with MLL translocation
(Daigle et al., 2011), our work suggests that DOT1L might be a
784, May 15, 2014 ª2014 Elsevier Inc. 781
Immunity
IL-22+ Cells Enhance Cancer Stemness
marker for colon cancer progression, and targeting this pathway
might be meaningful for colon cancer treatment.
EXPERIMENTAL PROCEDURES
Human Subjects
Patients diagnosed with colon carcinomas were recruited in the study. All
usage of human subjects in this study was approved by the local Institutional
ReviewBoard. One hundred fifty one formalin-fixed, paraffin-embedded tumor
tissue blocks were obtained during surgery. These patients underwent resec-
tion of the primary tumor at the SecondDepartment of General Surgery inMed-
ical University of Lublin between 2007 and 2008. The follow-up period was an
average 2.8 years. Additional 177 patients with colon cancer were evaluated
from the National Center for Biotechnology Information Gene Expression
Omnibus database (GSE17536) (Smith et al., 2010). Thirty-six fresh cancer tis-
sues were collected from patients with colon cancer newly diagnosed at the
University ofMichigan and theUniversity of Florida. Primary colon cancer cells,
immune cell subsets and all the in vitro and in vivo functional assays were per-
formed with single cells from fresh colon cancer tissues and peripheral blood.
Cell Isolation and FACS Analysis
Single cell suspensions were prepared from fresh colon cancer tissues as pre-
viously described (Curiel et al., 2004; Curiel et al., 2003; Kryczek et al., 2006;
Zou et al., 2001). Immune cells and tumor cells were enriched with paramag-
netic beads (StemCell Technology). Lin�CD45�EpCAM+ cells primary colon
cancer cells and CD4+CD45+ T cells were sorted from stained single cell sus-
pensions with a high speed cell sorter (FACSaria, Becton Dickinson Immuno-
cytometry Systems). Cell purity was >98% as confirmed by flow cytometry
(LSR II, BD). Cytokine profile was determined with intracellular staining and
analyzed by LSRII (BD).
Cell Culture and Sphere Formation
Three primary colon cancer cell lines (1, 2, 3) were established from fresh colon
cancer tissues. DLD1 and HT29 cell lines (ATCC) were used in the experi-
ments. Colon cancer cells were treated with recombinant IL-22 (R&D systems)
and/or colon cancer infiltrating CD4+ T cells for different time points. The
DOT1L inhibitor EPZ004777 (10 mM) and relevant antibodies were added in
conventional or sphere culture (Kryczek et al., 2012). Tumor cell sphere forma-
tion and gene expression were examined (Kryczek et al., 2012).
Lentiviral Transduction
Several lentiviral vectors were used to transduce colon cancer cells
and establish stable cell lines. The lentiviral transduction efficiency was
confirmed by GFP which was coexpressed by the lentiviral vector. The
knockdown efficiency was assessed by immunoblotting. The vectors
included pGIPZ lentiviral vector encoding gene-specific shRNAs for STAT3,
DOT1L, or scrambled shRNA (Puromycin resistant); lentiviral vectors encod-
ing an active form of Notch (the transmembrane and intracellular domains,
comprising residues 1,704–2,531) Notch-IC cDNA (Notch IC) (Wang et al.,
2011; Yamamoto et al., 2001), or constitutively active STAT3 domain
(STAT3C) (EF.STAT3C.Ubc.GFP) (Bromberg et al., 1999; Li and Shaw,
2006). Human DOT1L cDNA was cloned into the entry vector pENTR223.1
(Thermo Scientific Open Biosystem, OHS5894-202503093) and inserted
into the destination and expressing vector pDEST26 by Gateway� Recom-
bination Cloning Technique (Invitrogen). Human DOT1L expression vector
(pDEST26-hDOT1L) was verified by restriction enzyme digestion and DNA
sequencing.
Cytokine Detection
The amount of cytokines protein was detected either by ELISA (R&D) or flow
cytometry analyzer (FACS) as described previously (Curiel et al., 2004; Kryczek
et al., 2011). All samples were acquired with LSR II (BD) and analyzed with
DIVA software.
Real-Time RT-PCR
The mRNA was quantified by real-time RT-PCR. Specific primers are included
in the Supplemental Information (Table S5). SYBR GreenMaster Mix was used
782 Immunity 40, 772–784, May 15, 2014 ª2014 Elsevier Inc.
to detect fluorescence. Relative expressionwas calculated according to the Ct
value with normalization to GAPDH.
Immunoblot
Immunoblotting was performed with specific antibodies against human STAT3
(9132,Cell Signaling), phosphorylatedSTAT3 (9138,Cell Signaling),Oct3/4 (sc-
5279, Santa Cruz biotechnology), Nanog (ab21624, Abcam), Sox2 (MAB4343,
Millipore), H3K79me2 (ab3594, Abcam), H3K9me2 (ab1220, Abcam),
H3K9me3 (ab8898, Abcam), H4K20me3 (07-463, Millipore), H3K27me3
(07-449, Millipore), H3K36me3 (ab9050, Abcam), acetyl-Histone H3 (06-599,
Millipore), Histone H3 (9715, Cell Signaling), b-Actin (A5441, Sigma), Dot1L
(ab72454, Abcam), and Cleaved Notch1 (NICD, ab52301, Abcam). Signals
were detected by ECL reagents (GE Healthcare).
In Vitro and In Vivo Migration Assays
In vitro migration assay was performed in a Transwell system with a polycar-
bonate membrane of 6.5 mm diameter with a 3 mm pore size as described
(Curiel et al., 2004; Curiel et al., 2003). Purified T cell subsets were added to
the upper chamber and CCL-20 (5 ng/ml, R&D) was added to the lower cham-
ber. After 4 hr incubation at 37�C, the phenotype and number of T cells in the
upper and lower chambers was determined by FACS. In vivo migration assay
was performed in female NOD/Shi-scid/IL-2Rgnull (NSG) mice (6–8 weeks old,
Jackson Laboratory) (Curiel et al., 2004; Curiel et al., 2003; Kryczek et al.,
2012). Subcutaneous DLD-1 (106) tumor was established in NSG model.
Human T cell subsets (53 106) were treated with anti-CCR6 and isotype anti-
body and were intravenously transferred into these NSG mice. After 48 hr,
human T cells were analyzed in the tumors by FACS.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed according to the proto-
col with exceptions stated below (Upstate, Millipore; http://www.millipore.
com/techpublications/tech1/mcproto407). Crosslinking was performed with
1% formaldehyde or 1% paraformaldehyde for 10 min. To enhance cell lysis,
we ran the lysate through a 27 g needle three times and flash froze it in�80�C.Sonication was then performed with the Misonix 4000 water bath sonication
unit at 15% amplitude for 10 min. Protein and DNA complex was precipitated
with specific antibodies against H3K79me2 (abcam, ab3594), STAT3 (Santa
Cruz, SC-482), p300 (Santa Cruz, SC-585), and immunoglobulin G control
(Millipore). DNA was then purified using a DNA Purification Kit (QIAGEN).
ChIP-enriched chromatin was used for real-time PCR with SYBR Green
Master Mix, normalizing to input. Specific primers are listed in supplementary
information (Table S6).
Immunohistochemistry (IHC)
Immunohistochemical staining on colon cancer tissue sections was performed
on a DAKO Autostainer (DAKO,) with DAKO LSAB+ and diaminobenzadine
(DAB) as the chromogen. Serial sections of deparaffinized tissue sections
were labeled with rabbit polyclonal antibodies against human KMT4/Dot1L,
H3K79me2 and CCL20 (AbCam), or mouse anti-human VCAM1 antibody
(6G9, Abcam). H3K79me2 and DOT1L were localized in the nuclei and were
scored with the H-score method (Pirker et al., 2012). The H score is a method
of assessing the extent of nuclear immunoreactivity. The H score takes into ac-
count the percentage of positive cells (0%–100%) in each intensity category
(0–3+) and computes a final score, on a continuous scale between 0 and
300. The score is obtained by the formula: (33 percentage of strongly staining
nuclei) + (23 percentage of moderately staining nuclei) + (13 percentage of
weakly staining nuclei), giving a range from 0 to 300 (Pirker et al., 2012). Any
discrepancies were resolved by subsequent consultation with a diagnostic
pathologist. The tissues were divided into high and low H3K79me2 and
DOT1L expression based on the median value of H3K79me2 and DOT1L
expression per tissue section.
In Vivo Tumor Formation
Single cells were prepared from fresh colon cancer tissues. These colon can-
cer environmental cells contained all the primary cellular components in the
colon cancer environment including CD3+ T cells within CD45+ immune
cell population and lin�CD33�CD45�FSChiSSChi primary colon cancer cells.
These cells or colon cancer cells (102�5 3 106) were treated with anti-IL-22
Immunity
IL-22+ Cells Enhance Cancer Stemness
or IL-22, and were subcutaneously injected into dorsal tissues of NSG mice
(6–8 weeks old, Jackson Laboratory) (Curiel et al., 2004; Curiel et al., 2003;
Kryczek et al., 2012). Tumor size wasmeasured two times weekly with calipers
fitted with a Vernier scale. Tumor volume was calculated based on three
perpendicular measurements (Curiel et al., 2004; Curiel et al., 2003). Tumor
incidence was monitored.
Statistical Analysis
Wilcoxon rank-sum tests were used to compare two independent groups; for
paired groups, Wilcoxon signed rank tests were used for comparison. Corre-
lation coefficients (Spearman correlation, denoted by r, for ordinal data and
Pearson correlation, denoted by r, for continuous data), together with a p value
(null hypothesis is that r is in fact zero), were computed to measure the degree
of association between biomarkers. Log-rank test was used to compare time
to tumor initiation between two groups. Overall patient survival was defined
from date of diagnosis to disease related death. Data was censored at the
last follow-up for patients who were disease-free or alive at the time of anal-
ysis. Survival functions were estimated by Kaplan-Meier methods. Cox’s pro-
portional hazards regression was performed to model survival (all classified
as low and high based on the median value), after adjusting for age, grade
and stage. The adequacy of the Cox regression model was assessed with
graphical and numerical methods. All analyses were done with SAS 9.3
software. p < 0.05 was considered significant.
SUPPLEMENTAL INFORMATION
Supplemental Information includes six figures and six tables and can be found
with this article online at http://dx.doi.org/10.1016/j.immuni.2014.03.010.
AUTHOR CONTRIBUTIONS
I.K., Y.L., and W.Z. initiated the project. I.K., Y.L., N.N., and W.Z. designed the
experiments and wrote the paper. D.P., E.Z., L.V., and W.S. performed exper-
iments. I.K., N.N., and L.Z. analyzed data. Y.D., S.O., W.Z., M.M., G. W., J.F.,
and E.H. provided important intellectual and technical support, clinical speci-
mens, and clinical and pathological information. All authors read and approved
the manuscript.
ACKNOWLEDGMENTS
This work is supported (in part) by the National Institutes of Health grants
(CA123088, CA099985, CA156685, and CA171306 for W.Z.; CA142808 and
CA157663 for E.H.). We thank Wolf-Herve Fridman and Jay Hess for their in-
tellectual support and valuable discussions and Deborah Postiff, Michelle
Vinco, and Jackline Barikdar in the Tissue Procurement Core for their technical
assistance. We are grateful for Weisheng Wu and Yongsheng Bai in the Bio-
informatic Core for their help.
Received: October 23, 2013
Accepted: March 7, 2014
Published: May 8, 2014
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